A critical element of Pseudomonas aeruginosa pathogenesis is the ability to forms biofilms in the lungs of cystic fibrosis (CF) patients and on many other surfaces. Bacteria within biofilms produce one or more extracellular polymeric substances (EPS) that stabilize the biofilm and act as a scaffold. Alginate has been considered the major polysaccharide of the biofilm EPS matrix. However, recent studies indicate that alginate is not involved in the initiation of biofilm formation by non-mucoid P. aeruginosa strains, which are the first to colonize CF patients and are the cause of most acute P. aeruginosa infections. In CF patients, mucoid conversion typically occurs months or years after initial colonization. There remain significant gaps in understanding how P. aeruginosa survives the harsh, inflammatory-rich environment of the CF lung prior to converting to the alginate-producing phenotype. The central hypothesis to be examined is that P. aeruginosa has the capacity to express alternative EPS molecules that are essential for biofilm formation and persistence of P. aeruginosa during infection. This application will focus on one alternative polysaccharide, designated Eps1, which plays a critical role in biofilm formation. The overall objective is to determine the role of Eps1 in biofilm development, structure, resistance to antimicrobial agents, and P. aeruginosa pathogenesis. Aim 1 will focus on defining the genes within the eps1 gene cluster that are required for biofilm formation and understanding the role of Eps1 in formation of the biofilm matrix. In the second aim, the regulation of the eps1 gene cluster will be examined and the hypothesis that the eps genes are spatially and temporally controlled during biofilm development will be tested. Finally, experiments in aim 3 will determine if Eps1 contributes to the persistent phenotype of biofilms and is thus important in P. aeruginosa virulence. A further understanding of this critical biofilm component will lead to strategies aimed at inhibiting biofilm formation, which is a key aspect of P. aeruginosa pathogenesis.